Prevalence of Low-Frequency, Antiviral Resistance Variants in SARS-CoV-2 Isolates in Ontario, Canada, 2020-2023

This cohort study analyzes the prevalence of low-frequency SARS-CoV-2 variants in patient samples that could confer resistance to nirmatrelvir-ritonavir, a common antiviral medication.


Introduction
The COVID-19 pandemic has resulted in millions of deaths worldwide and continues to be an ongoing global public health challenge. Currently, nirmatrelvir-ritonavir is 1 of 2 oral antiviral medications for the treatment of SARS-CoV-2 and has reduced hospitalizations and deaths 1 ; yet, despite the substantial impact of antiviral medications on clinical outcomes, the development of antiviral resistance is an ongoing concern. Because SARS-CoV-2 is an RNA virus, the viral polymerase introduces errors in the genome during replication, and the virus exists as a quasi species in the patient. Therefore, it is unclear whether low-frequency variants may exist that could be selected for by drug pressure, as has been observed with other RNA viruses, such as influenza virus, 2 hepatitis C virus, 3 and HIV. 4 Nirmatrelvir-ritonavir inhibits the main viral protease encoded by the nsp5 gene, 5 and ritonavir acts as a boosting agent by inhibiting host cytochrome P450. Studies 6 have shown that viral rebound has occurred in a subset of treated patients, but it remains unclear whether this could lead to the selection of viruses with reduced antiviral susceptibility. Findings from in vitro studies 7 have identified variants with the potential to confer resistance to nirmatrelvir-ritonavir without affecting viral fitness.
Importantly, although these variants have been found in clinical isolates, 8 the prevalence of variants within the Global Initiative on Sharing All Influenza Data database of SARS-CoV-2 sequences was less than 1% among sequenced genomes. 9 However, this analysis was of the viral consensus sequence, and low-frequency allelic variants that could be selected for could not be detected. There has yet to be a comprehensive clinical evaluation of low-frequency variants that could be selected for during nirmatrelvir-ritonavir treatment. Next-generation sequencing allows for the detection of variants that are present at low frequency. This study examined the prevalence of low-frequency variants within SARS-CoV-2 sequences from clinical samples collected before and after the availability of nirmatrelvir-ritonavir in Ontario, Canada.

Study Design, Setting, and Samples
This cohort study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline, and did not require ethics board approval or informed consent because the study used deidentified viral sequences collected as part of routine surveillance work, in accordance with 45 CFR §46. This study was a descriptive retrospective analysis of SARS-CoV-2 samples that were submitted for routine diagnostic testing and sequencing. Samples were collected between March 1, 2020, and January 12, 2023, and analyzed at 4 laboratories with tertiary academic hospital centers in Ontario, Canada. All sequencing occurred less than 7 days from the time of collection. These laboratories serve multiple community hospitals, other academic tertiary care centers, as well as COVID-19 assessment centers, and have a total catchment of 5 million Ontarians.

Whole-Genome Sequencing
Samples that were positive for SARS-CoV-2 using reverse-transcription polymerase chain reaction testing with a cycle threshold less than 30 cycles underwent whole-genome sequencing, as has been previously described. 10 In a single center, the Illumina platform was used, and data consensus sequences and variants were called using FreeBayes. 11 The other 3 centers used the Oxford Nanopore Technologies platform, with Nanopore v3 Midnight primers using the native barcode, ARTIC SARS-CoV-2 bioinformatic protocol. 12 In this context, read length filtering of 400 bp to 700 bp and the V3 primer schema were used. For the Nanopore Midnight primers using rapid barcodes, the Medaka version of the ARTIC pipeline was used and validated alongside the ARTIC protocol. A read length filter of 150 bp to 2000 bp and the Midnight primer schema were used for the rapid barcoding protocol.

Statistical Analysis
Both platforms' sample analysis was then assessed for quality using ncov-tools software 13 version 1.9.1. A list of resistance variants was derived from data provided by the US Food and Drug Administration and a review of the literature. 7,8,14 Variant files for each sample were filtered to retain variants that were high quality (read depth Ն100 and quality score Ն30), had an allele frequency between 0.1 and 0.9, and were included in the variants of interest list. The nanopore analysis  (Table). Variation was found at only 33 of the residues identified by the US Food and Drug Administration as being associated with antiviral resistance (eTable in Supplement 1). Additionally, we did not observe more variation at residues that are known to interact with nirmatrelvir-ritonavir compared with other residues (Figure).

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Only 1 sample showed variation at residue H41, which is part of the catalytic dyad and is associated with drug binding. 15 Variation at T190, a residue that interacts with nirmatrelvir-ritonavir, was also noted in 1 sample. A total of 2 samples showed variants at M49 and N142, respectively, which have been associated with a decrease in activity of nirmatrelvir-ritonavir without a significant loss of protease activity. 16 No variants were seen at residues P252 or T304, both of which have been shown to reduce nirmatrelvir-ritonavir activity in vitro. 17 A total of 4 samples had low-frequency variants at L50; variants at this position have been shown to confer low-level resistance to nirmatrelvir-ritonavir. 7 A single isolate did show variation at T21, which is also associated with decreased antiviral susceptibility. 7

Discussion
Currently, nirmatrelvir-ritonavir is 1 of 2 oral antiviral treatments for SARS-CoV-2. This cohort study found that naturally occurring low-frequency variants in nsp5 are rare. Moreover, among the variants detected, no single variant predominated. Studies 18 suggest that only variants present at more than 15% are likely to represent viral adaptation in the face of selective drug pressure. The paucity of these variants suggests it may be difficult for resistant variants to be selected, and this may be a contributing factor to why resistance to nirmatrelvir-ritonavir has not yet been observed. A high degree of conservation of the viral protease is likely necessary because of its critical role in the viral

JAMA Network Open | Infectious Diseases
Prevalence of Low-Frequency, Antiviral Resistance Variants in SARS-CoV-2 Isolates in Ontario, Canada  Low-frequency variants identified by next-generation sequencing at key residues in the nsp5 gene that are associated with resistance to nirmatrelvir-ritonavir are shown. Frequency of detection is indicated by color.
a Denotes residues that have contact with nirmatrelvir-ritonavir.

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Prevalence of Low-Frequency, Antiviral Resistance Variants in SARS-CoV-2 Isolates in Ontario, Canada life cycle. 19,20 Interestingly, recent studies have estimated that the nonsynonymous variant rate for the protease is more than 10-fold lower than that for the viral polymerase. 9 Cell culture studies 7 identified the variants L50F and E166A as conferring resistance to nirmatrelvir-ritonavir, as well as T21I and T304I. Moreover, the presence of these variants in combination increased half maximal effective concentration values, without a complete loss of viral fitness 7 ; however, there appears to be a need for compensatory variants. 17 In our data, low-frequency variants at these positions were rare, and notably, combinations of low-frequency variants at multiple positions (eg, L50 and E166) were not detected. This suggests that there is a potential fitness cost to these variants that would not be observed in cell culture. The viral protease plays a role in moderating the host immune response, and these variants may affect that activity. This hypothesis is supported by our findings that low-frequency variants were found more often in regions outside the binding pocket and at sites that would be less likely to have a major effect on protease activity. Additionally, the use of nirmatrelvir-ritonavir has been relatively limited in Ontario because of an initially limited supply of the drug, government restrictions, and prescribing guidelines, 21 which could have limited the selective pressure placed on the virus and may partially account for the paucity of antiviral resistance variants observed.

Limitations
A limitation of this study is that variants detected at low frequencies could be due to artifacts of the sequencing process. Nonetheless, we included these data to highlight that even with this caveat, variations at these key positions are rare. Additionally, there were no clinical histories of the patients from which the samples were derived, and it cannot be discounted that some patients may have received antiviral treatment before testing. Also, patient samples with low viral loads were unable to be sequenced owing to assay limitations and, therefore, could not be included in our analysis. Future studies are needed that include relevant patient medical histories, as well as improved methods for sequencing of isolates from cases with low viral loads.

Conclusions
In conclusion, our data suggest that low-frequency variants of SARS-CoV-2 at the population level are rare. Surveillance efforts that involve sequencing of viral isolates should continue to monitor for novel resistance variants as nirmatrelvir-ritonavir is used more broadly.